Much work has been done in the dairy industry relating to the production of aroma. Aroma production in milk was considered in 1933 by Michaelian, Farmer, and Hammer, The Relationship of Acetylmethylcarbinol and Diacetyl to Butter Cultures, Iowa Agr. Expt. Sta., Research Bull. 155. This article reports that citric acid-fermenting bacteria could produce large amounts of aroma compound diacetyl. In 1941, Hoecker and Hammer investigated the ability of Streptococcus diacetilactis to produce aroma in butter. Flavor Development in Salted Butter by Pure Cultures of Bacteria, Iowa Agr. Expt. Sta., Research Bull. 290 (1941).
By 1961 Lundstedt had developed a procedure for enhancing the flavor of cottage cheese and other dairy products through the use of Streptococcus diacetilactis. (U.S. Pat. No. 3,048,490). Moseley, Elliker and Sandine developed a variation on the Lundstedt process for making cottage cheese by 1964 (U.S. Pat. No. 3,323,921). In 1970, a still further variation of the process of making cottage cheese was developed by Sing (U.S. Pat. No. 3,968,256). This process involved the bulk addition of concentrated Streptococcus diacetilactis to cottage cheese without the need for further incubation. In all of these processes, one of the objects was to use Streptococcus diacetilactis because it produced diacetyl which enhanced the flavor.
It was also observed in the early 60's that the presence of Streptococcus diacetilactis had an inhibitory effect on spoilage organisms. In order to achieve the maximum inhibition of spoilage organisms, a relatively high concentration of Streptococcus diacetilactis in the finished cheese would be desired. However, if such a large concentration of Streptococcus diacetilactis were used, there was a tendency for excess production of the flavor compounds of diacetyl and acetaldehyde. Thus with the known processes of the prior art, one could obtain either an ideal maximum shelf life or an ideal flavor production, but not both. Efforts to overcome these problems lead to attempts to isolate strains of bacteria which had less flavor but which retained the full inhibitory effect.
In 1969, Burrow, Sandine, Elliker and Speckman pointed out the problem of too much flavor when maximum shelf life was obtained. Characterization of Diacetyl Negative Mutants of Streptococcus diacetilactis, Journal of Dairy Science, Vol. 53, p. 121-125. They noted that there was a need for strains of S. diacetilactis with impaired abilities to synthesize diacetyl. It was suggested in this 1969 article that since acetaldehyde appears to be a precursor of diacetyl during citrate metabolism, it was also believed that mutants of S. diacetilactis could be isolated with reduced aldehyde production. The authors reported that "Such strains would prove useful in manufacturing cultured products which often suffer from the green flavor defect." The authors reported that mutants of Streptococcus diacetilactis which they had developed produced an average of forty percent more lactic acid than parent strains; had acetaldehyde production which varied from quantities equal to the wild type to less than one-third the amount produced by the parents; and which retained inhibitory powers against food spoilage bacteria similar to their respective parents. However, the high acid and acetaldehyde production by the isolated mutants made their use in cottage cheese dressing impractical. The article suggested that this approach to extending the shelf life of cottage cheese and other foods seemed promising and reported that efforts were being continued to isolate new mutants which would produce less acid and aldehyde and still yield high cell numbers.
While efforts along this line had met with failure and research along this line had apparently ended, research concerning the biochemical pathways of Streptococcus lactis and Streptococcus diacetilactis has been taking place. In work of Kempler and McKay, studies of plasmid activity within Streptococcus diacetilactis led to the generation of various mutant forms of Streptococcus diacetilactis. Two articles about this work are entitled "Characterization of Plasmid Deoxyribonucleic Acid in Streptococcus lactis Subsp. diacetilactis: Evidence for Plasmid-Linked Citrate Utilization", Applied and Environmental Microbiology, Feb. 1979, pp. 316-323, Vol. 37, No. 2 and "Genetic Evidence for Plasmid-Linked Lactose Metabolism in Streptococcus lactis Subsp. diacetilactis", Applied and Environmental Microbiology, May, 1979, pp. 1041-1043, Vol. 37, No. 5. In studying the mechanism of the production of diacetyl from citrate as well as the lactose metabolism of Streptococcus diacetilactis, the authors Kempler and McKay treated S. diacetilactis strains 18-16 and DRCl with acridine orange to eliminate various ones of the plasmids which were within the bacteria. As early as 1972, McKay et al. showed that acriflavin treatment of S. diacetilactis 18-16 resulted in the appearance of lactose-negative derivatives, implying the involvement of plasmid DNA in lactose utilization. While innumerable mutants were developed during the 70's for the purpose of determining the biological mechanisms of the bacteria, there were no reports of Kemper and McKay over the retention or lack of retention of inhibitory powers against food spoilage bacteria. Indeed, most of the mutant bacteria only had value in the research setting since they often had commercially desirable metabolic pathways significantly damaged by the mutating procedure. Thus the typical mutant developed for purposes of understanding the metabolic pathways was not considered as a bacterium which had commercial application. Most mutants of this type would be totally unsuited for commercial use.